The extensive research and development taking place in the rapidly growing fields of molecular biology and biotechnology has increased the demand for ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and proteins which can be used in experimental work and/or commercial production. All biological tissue contains RNA, DNA and proteins; however, for many clinical and research applications, it is necessary to isolate and characterize these components from tissue and cell cultures. For example, RNA and DNA isolation is necessary for molecular cloning of genes which are important in the medical and agricultural fields (e.g., insulin, growth hormone genes, and genes responsible for increased plant productivity). In addition, isolation and characterization of RNA, DNA and proteins is necessary for the early detection of molecular defects in the human genome, and for gene expression studies. In many contexts it is necessary or desirable to isolate the RNA, DNA and proteins from the same biological sample. This is extremely difficult, if not impossible, when the tissue sample size is very small, for example in biopsies, and cannot be divided and used in separate isolation techniques for each component.
There are a variety of known techniques for isolating RNA from biological tissue. For example, my earlier U.S. Pat. No. 4,843,155, the content of which is expressly incorporated herein by reference, discloses an efficient product and process for isolating RNA from biological tissue samples. Utilizing the product and process of the '155 patent, RNA can be isolated in about four hours. The '155 patent refers to and describes several other known techniques for isolating RNA, each of which has specific drawbacks. Additionally, neither the process of the '155 patent, nor the other processes referred to therein are directed to simultaneously isolating RNA, DNA, and proteins from the same tissue sample.
Other techniques are known for the simultaneous isolation of RNA and DNA. For example, U.S. Pat. No. 5,010,183 discloses a process for purifying DNA and RNA from a variety of sources, including cells, cell lysates, viruses, tissues, blood and other body fluids, employing a cationic detergent to complex with the nucleic acids. Additional protocols for simultaneous isolation of RNA and DNA are described in the following articles: Chan, V. T.-W., et al.: Anal. Biochem., 168, 16-24 (1988); Nicolaides, N. C., et al.: BioTechniques, Vol. 8, No. 2, 154-156 (1990); and Raha, S., et al.: Gene Anal. Techn. (7), 173-177 (1990). Each of the described protocols extract RNA and DNA using an extraction solution containing detergents (e.g., NONIDET P-40, sodium lauryl sulfate, and sodium lauryl sarcosine) and phenol supplemented with RNase inhibitors.
A procedure for simultaneously isolating DNA, RNA and protein from the same biological sample is described in Coombs, L. M., et al.: Anal. Biochem., 188, 338-343 (1990). This method is a modification of the Chirgwin et al. method referred to in my U.S. Pat. No. 4,843,155. In summary, this method is performed as follows: cell or tissue samples are homogenized in 4M guanidinium. The homogenate is overlayered on a cesium chloride (CsCl) gradient. Following centrifugation for 18 hours at 110,000-150,000 g, the top guanidinium phase contains proteins, the upper layer of CsCl contains DNA, and RNA collects at the bottom of the ultracentrifuge tube. Purification of the RNA, DNA and protein fractions collected from the cesium chloride gradient is completed in the next 12-24 hours. This method is disadvantageous because it is extremely time consuming (on the order of 2-3 days to complete) and because it requires an ultracentrifuge, which is an expensive piece of equipment that can process only a limited number and size of samples simultaneously.
Thus, there is a definite need for an efficient and accurate method for the simultaneous isolation of RNA, DNA and proteins from the same biological sample.